Abstract Despite extensive drug discovery efforts targeting G-protein coupled receptors (GPCRs), these important targets for drug discovery remain challenging proteins for direct, on-target drug discovery campaigns. To answer the challenge posed by GPCRs, Biodesy will use its Biodesy Delta device, a platform based on Second Harmonic Generation (SHG) that allows one to measure changes in biomolecule conformation upon ligand addition in real-time, to develop a commercial screening assay for identifying compounds that directly modulate the structure and function of GPCRs. SHG is a non-linear optical process that is highly sensitive to orientational changes of a SH-active, 370Da dye probe, covalently attached to a protein of interest at either lysine or cysteine residues. The technology requires 2-10pmol target protein per well and is operated in a 384- or 1536- well microtiter plate format allowing for the collection of tens of thousands of data points per day. Unlike other optical screening methods, that have been tried and require fusion proteins, the SHG dye can be added without altering cellular expression, activity of the targeted receptor, and affinity for receptor ligand. Using SHG, Biodesy has measured a range of molecular interactions between target proteins and small molecules, fragments, peptides and antibodies. To enable screening of GPCRs, we have successfully developed a prototype non-lipid surface that allows tethering of GPCRs and is resistant to detergents used to solubilize and stabilize membrane protein samples in solution. The new plate surface demonstrated minimal non-specific binding of the purified labeled NTR1 prototypical GPCR. We have also demonstrated that SHG dye labeled NTR1 tethered to the surface remains functional by examining the binding of two control ligands to the tethered protein. Additional changes to the current components of our Biodesy Delta device and screening plates can create a commercial kit that detects and analyzes SHG signals from dyes attached to a GPCR of choice. In this SBIR phase I project, we will identify and optimize the surface coating conditions of multi well plates that will enable SHG assays with tethered GPCRs. Then, we will explore different labeling positions within a GPCR (NTR1 receptor) for determination of the optimal labeling location for discriminating small molecule GPCR agonists from antagonists. The final step will be to integrate the new GPCR-compatible multi- well plates and the optimally labeled proteins into a ligand screening procedure. To test the ability of the assay to differentiate between different types of ligands, it be tested against an expanded set of known agonists and antagonists as well as compounds with unknown mechanisms of action. This test will validate and demonstrate the robustness of the assay and its ability to predict Mechanism of action of GPCR ligands.